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Virus electrodes address two major challenges associated with biosensing. First, the surface of the viruses can be readily tailored for specific, high affinity binding to targeted biomarkers. Second, the viruses are entrapped in a conducting polymer for electrical resistance-based, quantitative measurement of biomarker concentration. To further enhance device sensitivity, two different ligands can be attached to the virus surface, and increase the apparent affinity for the biomarker. In the example presented here, the two ligands bind to the analyte in a bidentate binding mode with a chelate-based avidity effect, and result in a 100 pM experimentally observed limit of detection for the cancer biomarker prostate-specific membrane antigen. The approach does not require enzymatic amplification, and allows reagent-free, real-time measurements. This article presents general protocols for the development of such biosensors with modified viruses for the enhanced detection of arbitrary target proteins.

The article discusses phage display mechanisms and phage-based biosensors. Topics include the: advantages of using the bacteriophage called filamentous phage; manipulation of polypeptides on virus particle surfaces; selection of a polypeptide receptor and affinity screening; methods for immobilizing filamentous phage on biosensor surfaces; antibody binding on a covalent virus surface; prostate-specific membrane antigen--a cancer biomarker; biosensor transducer--electrochemical impedance spectroscopy; and binding of target molecules to macroscopic gold electrodes.

The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnosis and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. This report describes a bioaffinity matrix of viruses integrated into PEDOT films for electrochemical sensing of prostate-specific membrane antigen (PSMA), a prostate cancer biomarker. High sensitivity to PSMA resulted from synergistic action by two different ligands to PSMA on the same phage particle. One ligand was genetically encoded, and the secondary recognition ligand was chemically synthesized to wrap around the phage. The dual ligands result in a bidentate binder with high-copy, dense ligand display for enhanced PSMA detection through a chelate-based avidity effect. Biosensing with virus–PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification. [ABSTRACT FROM AUTHOR]

We demonstrate the de novo fabrication of a biosensor, based upon virus-containing poly(3,4-ethylene-dioxythiophene) (PEDOT) nanowires, that detects prostate-specific membrane antigen (PSMA). This development process occurs in three phases: (1) isolation of a M13 virus with a displayed polypeptide receptor, from a library of ≈1011 phage-displayed peptides, which binds PSMA with high affinity and selectivity, (2) microfabrication of PEDOT nanowires that entrain these virus particles using the lithographically patterned nanowire electrodeposition (LPNE) method, and (3) electrical detection of the PSMA in high ionic strength (150 mM salt) media, including synthetic urine, using an array of virus-PEDOT nanowires with the electrical resistance of these nanowires for transduction. The electrical resistance of an array of these nanowires increases linearly with the PSMA concentration from 20 to 120 nM in high ionic strength phosphate-buffered fluoride (PBF) buffer, yielding a limit of detection (LOD) for PSMA of 56 nM. [ABSTRACT FROM AUTHOR]

A dense virus layer, readily tailored for recognition of essentially any biomarker, was covalently attached to a gold electrode surface through a self-assembled monolayer. The resistance of this ‘virus electrode’, ZRe, measured in the frequency range from 2 to 500 kHz in a salt-based pH 7.2 buffer, increased when the phage particles selectively bound either an antibody or prostate-specific membrane antigen (PSMA), a biomarker for prostate cancer. In contrast to prior results, we show the capacitive impedence of the virus electrode, Zlm, is both a noisier and a less sensitive indicator of this binding compared to ZRe. The specificity of antibody and PSMA binding, and the absence of nonspecific binding to the virus electrode, was confirmed using quartz crystal microbalance gravimetry. [ABSTRACT FROM AUTHOR]